Synthetic mammalian expression circuits which enable reversible and adjustable transgene expression have been essential for recent advances in functional genomic research, drug discovery, manufacturing of difficult-to-produce protein therapeutics, the design of synthetic gene networks replicas reaching the complexity of electronic circuits and gene therapy applications.
To date, a multitude of heterologous transgene expression systems, for use in mammalian cells and transgenic animals, have been described (Weber W., Fussenegger M., 2007, Curr Opin Biotechnol 18(5):399-410). The prevailing design consists of a heterologous small molecule-responsive transactivator engineered by fusing a prokaryotic repressor to an eukaryotic transactivation domain and a transactivator-specific promoter containing the matching prokaryotic operator linked to a minimal eukaryotic promoter. Inducer-triggered modulation of the transactivator's affinity to its cognate promoter results in adjustable and reversible transcription control of the specific target gene. In recent years, a panoply of such heterologous transcription control modalities have been developed which are responsive to a variety of inducer molecules such as antibiotics, steroid hormones and their analogs, quorum-sensing molecules, immunosuppressive and anti-diabetic drugs, biotin, L-arginine as well as volatile acetaldehyde (WO2005/021766). Apart from gaseous acetaldehyde which can simply be inhaled, all other inducers need to be either taken up orally or be administered by injection in any future gene therapy application. Transdermal and topical delivery of inducer molecules, which would provide advantages over conventional injection-based or oral administration such as convenience, improved patient compliance and elimination of hepatic first-pass effect, have not yet been developed.
Phloretin, which is a natural plant defense metabolite with antibacterial activity (Teran W., Krell T., Ramos J. L., Gallegos M. T., 2006, J Biol Chem 281(11):7102-7109), occurring in the root bark of apple trees as well as in apples, has been studied as a possible penetration enhancer for skin-based drug delivery, attenuates inflammation by antagonizing prostaglandins, protects the skin from UV light-induced photodamage and is evaluated as a chemopreventive agent for cancer treatment. Since the plant rhizosphere is one of the natural habitats of Pseudomonas putida (strain DOT-T1E), this prokaryote has evolved the RND family transporter TtgABC with multidrug recognition properties which is controlled by its cognate repressor TtgR binding to a specific operator, (OTtgR) in the TtgR promoter (PTtgR). Phloretin has been shown to bind to the TtgR-operator complex at a stoichiometric ratio of one effector molecule per dimer of TtgR and to release TtgR from OTtgR which results in induction of TtgABC production and effective pump-mediated efflux of the flavonoid from P. putida (Teran W. et al., loc. cit.).